Natural Killer (NK) cell deficiency (NKD) is a recently appreciated subset of primary immunodeficiency (PID) in which NK cell deficits represent the main immunological defect. These diseases are typically severe; patients suffer from profound and often lethal susceptibility to viral infection and cancer. To date, only three NKD genes have been identified and little is known of the biological mechanisms by which they interfere with NK cell numbers and/or function. At the same time, no good therapeutic options are available for those affected. For over a decade, our laboratory has sought genetic, immunologic, and therapeutic solutions to these rare, but deadly diseases. In the process, we have established a robust worldwide referral base, assembled an expert team of geneticists, immunologists and clinicians, and are now poised to dramatically advance our understanding of NKD and the role of NK cells in health and disease. Through two Aims, we will define genes responsible for human NKD (Aim 1), and determine their relevance through intensive biological characterization (Aim 2). In Aim 1, we use whole exome sequencing and tandem chromosomal microarray to identify NKD mutations in the world's largest cohort of NKD patients. We have already identified 7 novel gene candidates using this approach. In Aim 2, we will rigorously evaluate our candidate NKD genes, defining their roles in NK cell cytolytic functions, developmental maturation, receptor repertoire, and immunoregulatory and inflammatory function using a carefully thought out progression of cutting-edge immunologic experiments. Tools to be used include gold-standard 51Cr-release killing assays, Western blots, shRNA knockdown, gene knockout using CRISPR/Cas technology, extended multiparameter flow cytometry-based phenotypic and functional analysis, high- and super-resolution quantification of the lytic immunological synapse, imaging flow cytometry quantification of transcription factor activation and lytic synapse dynamics, and in vitro NK cell differentiation from CD34+ hematopoetic stem cells. Specifically, we will prove that these genes cause NKD in ex vivo NKD patient cells, ex vivo healthy donor NK cells and NK cell lines (knock-outs and knock-ins), and patient-derived induced pluripotent stem cells (iPSC) and reconstituted humanized mice. Additionally, the potential therapeutic benefit of clinically available cytokines will be evaluated for each candidate. Overall our approach represents iterative cycles of genetic discovery, confirmation, biological validation, and immunologic insight. We are confident that these innovative approaches, combined with our established NK cell and genetic expertise, and privileged NKD patient pipeline, will drive meaningful diagnostic, therapeutic, and basic science progress in this burgeoning field. We provide preliminary data from two examples of our discoveries, ATP6V0A2 and MCM10, as concrete examples of how the proposed work will substantively advance the field.